Cell-free Translation Systems
457
an RNA ligase. If unnatural amino acids
are good substrates for aminoacyl tRNA
synthetases, they can be attached to tRNA
by an appropriate aminoacyl tRNA syn-
thetase. The unnatural amino acid ligated
to a suppressor tRNA is further subjected
to the cell-free translation system and the
codon-forming base pairing with the an-
ticodon of suppressor tRNA is translated
into a proteinogenic amino acid.
Although the incorporation of an un-
natural amino acid into a polypeptide
using cell-free translation is highly advan-
tageous compared to an
in vivo
system,
there are several problems to be solved.
Oneprob
lemistheaminoacy
lat
ionofthe
suppressor tRNA after one reaction cy-
cle of translation. The suppressor tRNA
recharged by an aminoacyl tRNA syn-
thetase during cell-free translation inserts
a canonical amino acid at the amber codon,
which hinders the efFcient incorporation
of unnatural amino acid. To solve this
problem, tRNAs that are not recognized
by an endogenous aminoacyl tRNA syn-
thetase has been developed. ±or instance,
taking advantage of the fact that yeast
tRNA
Tyr
and tRNA
Gln
do not serve as a
substrate for the
E. coli
cognate aminoa-
cyl tRNA synthetase, the suppressor tRNA
having the framework of yeast tRNAs is
utilized as an adaptor tRNA for unnatural
amino acids in an
E. coli
cell-free transla-
tion system to prevent recycling of tRNA.
E. coli
tRNA
Tyr
, which is not recognized
by the eukaryotic tyrosyl-tRNA synthetase,
can be utilized in wheat germ extract and
vice versa
. Another problem is that the pre-
sence of R±-1 corresponding to the amber
codon in the extract caused the termina-
tion to produce an incomplete peptide,
reducing the percentage of peptide con-
taining unnatural amino acid. Using the
cell-free translation system in which all
the components are in a puriFed state,
this competition is circumvented by elim-
inating R±-1 from the system, achieving
almost full suppression efFciency.
Another problem in the amber suppres-
sors
tra
tegyistha
ton
lyonecodoninthe
open reading frame is available for the
introduction of unnatural amino acids.
To allow the incorporation of several va-
rieties of unnatural amino acids into a
single polypeptide, the four-base codon
method was developed. In this approach,
tRNA having a four-base anticodon is syn-
thesized by transcription reaction using
RNA polymerase, and chemically aminoa-
cylated, followed by cell-free translation. By
the four-base codon–anticodon method,
one is enabled to incorporate multiple
unnatural amino acids at plural codons.
Obviously, due to competition with en-
dogenous tRNAs, incorporation of an
unnatural amino acid into the polypeptide
never reaches full yield, but by employ-
ing the four-base codons competing with
minor three-base codons, the efFciency
of incorporation is easily improved, de-
pending on the amount of endogenous
tRNA. This artiFcial genetic code can be
expanded by exploiting the Fve-base anti-
codon tRNA.
The expansion of the genetic code in
cell-free translation was challenged by
the utilization of unnatural nucleobases
in codon and anticodon correspondence.
The utilization of one additional artiFcial
base pairing besides A-U and G-C pairing
in the translation process is able to, in
principle, extend the canonical 64 codons
to 216 (6
×
6
×
6) codons in the genetic
code, which gives rise to a vast possibility
for the incorporation of various unnatu-
ral amino acids into polypeptides. ±irst,
isoCandisoG(±ig.3a)wereintroducedin
the codon and anticodon, respectively, and
the resultant artiFcial codon corresponded
to the unnatural amino acid. Recently,
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